Conjugative transposons carry determinants of antibiotic resistance in Gram-positive bacteria. They are remarkably promiscuous, and thus contribute to the spread of resistance among important human pathogens. Because of their ability to transfer between many different bacterial species, they have become important tools for the genetic analysis of a wide variety of bacteria. During transposition, the transposon excises from the donor DNA molecule to form a circular intermediate which then can recombine with the target DNA, resulting in insertion of the transposon into a new site. Two transposon-encoded proteins are required for or implicated in the recombination reactions that lead to transposon excision and integration. The DNA sites and proteins involved in transposition bear similarities to the sites and proteins involved in bacteriophage lambda site-specific recombination. However, analysis of the circular transposon intermediate and of the recombination mechanism involved in conjugative transposition, binding sites for the two transposon-encoded proteins will be identified and characterized in vitro and in vivo. Host target sequences will be characterized in detail to understand the nature of preferred target sequences. Transposon proteins will be characterized biochemically. Finally, transposon and host functions that affect transposition in different hosts will be identified. It is anticipated that a molecular analysis of conjugative transposition will reveal a new mechanism of recombination, with important implications for understanding both transposition and nonhomologous recombination.